RU2765736C2 - Hyper-stabilized liposomes increasing directed targeting of mitotic cells - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to the field of medicine, namely to hyper-stable liposome for the treatment of cancer, containing an internal medium separated from an external medium by membrane, where the internal medium contains an antimitotic drug, two cations enclosed in the internal medium, where the antimitotic drug is a polo-like kinase inhibitor, where membrane contains 1,2-distearoyl-sn-glycerin-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSFE-PEG2000), hydrogenated egg L-α-phosphatidylcholine (HEPC), and cholesterol (Chol), where the molar ratio of HEPC:Hol:DSFE-PEG2000 is 50:45:5, where two anions are citrate and phosphate, citrate and acetate or acetate and phosphate in a molar ratio of 1:3 or 1:4, and where the enclosed antimitotic drug has a slow release rate from hyper-stable liposome, which is quantitatively less than 0.6% in 12 hours or less than 5% in 8 days, where hyper-stable liposomes are suspended in 600 mM sucrose, it also relates to a liposomal composition for the treatment of cancer, containing hyper-stable liposome in an aqueous medium and to a pharmaceutical composition for the treatment of cancer containing hyper-stable liposome and a pharmaceutically acceptable excipient, it also relates to hyper-stable liposome for use as a drug for the treatment of cancer in a subject.

EFFECT: group of inventions provides for the creation of liposomes that maximize the temporary effect of an agent inhibiting mitosis in order to increase the proportion of dividing cancer cells that can be affected by the agent inhibiting mitosis.

15 cl, 2 tbl, 14 dwg, 5 ex

Description

BACKGROUND OF THE INVENTION

This invention relates to the field of cancer treatment. More specifically, the invention relates to hyperstable liposomes for use in the treatment of cancer and methods of treating cancer using hyperstable liposomes.

Publications and other materials used here to illuminate the technical field of the invention or obtain additional details regarding the practice are included here by reference, and for convenience are listed in the Bibliography, respectively.

If cancer is basically a state of excessive cell division, then mitosis-regulating enzymes should be excellent anti-cancer drug targets. Indeed, it was the success of microtubule-targeting agents (AMTs) as a class of drugs that prompted the search for more specific ways to inhibit mitosis, which made it possible to avoid the peripheral neuropathy associated with MTs [1-3]. Enzymatic regulators that play a key role in mitosis, such as Polo-like kinases (PLKs) [4,5], kinesin spindle protein (KSP) [6,7], and aurora kinases [8,9] immediately became high priorities. drug targets. However, despite over US$10 billion spent on the development of 25 mitosis-specific agents, efficacy has been low and clinical efficacy has not been reported [10,11].

However, mitosis-regulating enzymes can be inherently poor drug targets because only a limited proportion of tumor cells are actually dividing at any given time. As summarized by Komlodi-Pasztor et al [10], “for targeted therapy to be effective, the target must be present.” This argument implies, however, that mitosis-regulating enzymes may still be effective if tumor bioavailability can be temporarily maintained. The fact that preclinical testing of the PLK inhibitor BI 2536 only showed tumor shrinkage when administered twice a week [4] supports the idea that sustained bioavailability increases the likelihood of tumor cell capture during cell division.

Liposomes are well known colloidal particles that are used for drug delivery. It is well known that small molecules, including drugs, can be "remotely loaded" into liposomes by creating a physicochemical differential between the internal and external environment of the liposome [16-18]. It is important to note that the drug must be able to permeate the membrane into the external environment, but become charged and therefore be trapped by diffusion into the internal environment. If the drug is a weak base, one way to create this differential is to encapsulate the buffer anions in the interior of the liposome, which creates a low pH relative to the environment.

It is known that liposomes use the porosity of the tumor endothelium for access and survival in tumor tissues [12, 13]. This phenomenon, called the Enhanced Permeability and Retention (APR) effect, was first demonstrated with doxorubicin, resulting in the liposomal anti-cancer drug Doxil™ [14,15]. It turned out that the stability of liposomal encapsulation is a double-edged sword, as demonstrated by Doxil™. On the one hand, the effect of the drug on healthy tissue is reduced. On the other hand, the slow leakage of doxorubicin from liposomes inhibits efficacy because most anticancer drugs require high tumor concentrations to be effective.

In contrast to doxorubicin, BI 2536 is effective at 1000x concentration of doxorubicin, implying that long exposure rather than maximum concentration should greatly enhance efficacy.

It is desirable to develop systems that maximize the temporal exposure of the mitosis inhibitory agent in order to increase the proportion of dividing cancer cells that can be affected by the mitosis inhibitory agent.

SUMMARY OF THE INVENTION

This invention relates to the field of cancer treatment. More specifically, the invention relates to hyperstable liposomes for use in the treatment of cancer and methods of treating cancer using hyperstable liposomes.

Thus, in one aspect, the present invention relates to hyperstable liposomes encapsulating an anti-mitotic drug. In some embodiments, the antimitotic drug is BI 2536, Ispinesib (SB 715992), MK 0457 (VX 680), AZD 1152, PHA 680632, PHA 739358, MLN8054, MLN8237, R763, AT9283, SNS 314, SU 6668, ENMD 2076 BI 811283, CYC116, ENMD 981693, MKC 1693, ON01910, GSK 461364, HMN 214, BI 6727, SB 743921, MK 0731, or ARRY 520. In some embodiments, any suitable liposomal moiety may be used to prepare hyperstable liposomes. In some embodiments, hyperstable liposomes are spatially stabilized. In some embodiments, hyperstable liposomes are derived from a fat blend containing HFPC:Chol:DSPE-PEG2000 (HPC: hydrogenated egg L-α-phosphatidylcholine; Chol: cholesterol; 3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] in a molar ratio of 50:45:5 In some embodiments, hyperstable liposomes contain an internal environment having one or more anions, preferably two or more anions, which is designed to slow isolation of the antimitotic agent from hyperstable liposomes.In some embodiments, one or more anions, preferably two or more anions, may be as described herein.In some embodiments, the internal environment contains one or more cations.In some embodiments, one or more cations can be as described here The best combination of anions and cations can be easily determined for a given anti-mitosis drug using the methods described here.

In some embodiments, a pharmaceutical composition is provided that contains the hyperstable liposomes described herein, with or without at least one pharmaceutically acceptable excipient and/or carrier. Suitable pharmaceutically acceptable excipients and carriers are well known in the art.

In a second aspect, the present invention provides a method for treating cancer using the hyperstable liposomes described herein. According to this method, a therapeutically effective amount of hyperstable liposomes is administered to a patient, eg a human, in need of treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

In FIG. 1 shows a procedure for studying the ability of BI 2536 to separate from various saline solutions into hexanol.

In FIG. 2 shows the relative fluorescence of BI 2536 extracted into hexanol from various solutions of one anionic salts using the procedure described in FIG. 1. The separation of BI 2536 to hexanol and saline depends on the identification and concentration of the anion of the salt. The abbreviations used are citrate (C), acetate (A), phosphate (P), 2-(N-morpholino)ethanesulfonate (M), and hydrochloric acid (H). All saline solutions are 0.8M and adjusted to pH 3 with sodium as the cation. Error bars represent standard errors.

FIG. 3 shows the relative fluorescence of BI 2536 extracted into hexanol from various anion pairings using the technique described in FIG. 1. Adjusting the molar ratios of anion pairings can affect the separation of BI 2536 to hexanol. All salts are 0.8 M and adjusted to pH 3 with sodium as the cation. Individual salts have a concentration of 0.8 M. The number in front of the abbreviation indicates a concentration other than 0.8 M of the total salt concentration.

In FIG. 4 shows release rates related to EC ₅₀ for liposomal BI2536 but not liposomal Doxorubicin. A continuous array of drug release rates was created using single and pairwise combinations of the following anions to perform gradient loading: citrate (C), acetate (A), phosphate (P), 2-(N-morpholino)ethanesulfonate (M), and hydrochloric acid (H). Abbreviations of double letters mean paired combinations of anions, each in half of the total concentration. Scatter plots of cytotoxicity (EC ₅₀ ) versus release rates for BI 2536 (top) and Doxorubicin (bottom) are shown for hypotonic (aqueous) and hypertonic conditions (sucrose). The bar chart shows EC ₅₀ to compositions ranked by release rates for the same data. The dotted lines for all plots show the EC ₅₀ of the unencapsulated drug. Release rates are based on the amount of drug released after 12 hours of incubation. Spearman's rank correlation ( r _s ) and corresponding p-values are presented.

In FIG. 5A and 5B show the potency of liposomal BI 2536 adjusted for the citrate:phosphate ratio setting. In FIG. 5A: Shows scatter plots of EC ₅₀ versus release rate measured on days 3 and 8 for various citrate:phosphate (C:P) ratios. In FIG. 5B: Mice with xenograft HCT116 colorectal cancer cells are treated with a single dose of liposomal BI 2536 formulated at various C:P ratios. 3 mice are used in each experimental direction. Volumes and masses of tumors are presented. Error bars represent standard errors.

In FIG. 6A and 6B show the in vivo efficacy and toxicity of liposomal BI 2536 in mice with HCT116 xenografts. Tumor volumes and masses and Kaplan-Meier survival curves are shown for single dose treatment on day 0 (FIG. 6A) or two doses on days 0 and 7 (FIG. 6B). All liposomal BI 2536 treatments were formulated with various citrate:phosphate (C:P) ratios as indicated and administered at a dose of 340 mg/kg after accounting for encapsulation efficacy. Free BI2536 is administered at a maximum tolerated dose of 100 mg/kg. Error bars represent standard errors. Tumor volumes differed significantly ( p <0.05) for hyperstable liposomes (C:P=1:3)) and other groups starting on day 9 for one dose and starting on day 14 for two doses. Mice treated with C:P(1:3) survived significantly longer ( p <0.05) than the other groups. The Kaplan-Meier curve shows the percentage of survival over time. Checkmarks indicate deaths. The difference between BI-L2C6P and all other treatments was significant, Mantel-Cox log-ranked p -values are presented for survival curves, showing that mice treated with C:P(1:3) survived significantly longer and for one ( p = 0 .0164) and for two ( p =0.0349) doses.

In FIG. 7A-7D show the pharmacokinetic distribution and bioavailability of BI 2536 after treatment with hyperstable lipsomic BI 2536. (FIG. 7A): HCT116 xenograft-bearing mice are treated with BI 2536 encapsulated using various citrate:phosphate ratios. Each data unit includes 3 mice. BI 2536 is extracted from tissues at various time points quantified by fluorometry. Data units and error bars represent mean and standard errors, respectively. Significant differences ( p <0.05) between hyperstable liposomes (C:P=1:3) and other groups are shown with asterisks. (FIG. 7B): Shows tissue exposure to BI 2536 as measured by area under the curve. (Fig. 7C): shows the percentage of mitotically suppressed cells at 1.5 and 5.5 days after treatment. Each bar is derived from 6 separate visual fields of 2 non-adjacent H&E colored sections. Error bars represent standard errors. (FIG. 7D): exemplary H&E images are shown for various treatments. Arrows indicate examples of mitotically suppressed cells. Scale 10 µm.

In FIG. 8A and 8B show the in vivo efficacy of liposomal BI 2536 in HCT116 xenograft mice. Tumor volumes are shown for single dose treatment on day 0 (FIG. 8A) with liposomes formulated at different citrate:acetate ratios and (FIG. 8B) liposomes formulated at different citrate:acetate ratios but with sodium cation replaced by ammonium. All formulations were administered at a dose of 340 mg/kg BI 2536. Error bars represent standard errors.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to the field of cancer treatment. More specifically, the invention relates to hyperstable liposomes for use in the treatment of cancer and methods for treating cancer using hyperstable liposomes. Extreme prolongation of the release of a mitosis-inhibiting drug from hyperstable liposomes has been found to improve the efficacy of cancer treatment by increasing the proportion of cancer cells affected. Slow release of the mitosis-inhibiting drug from hyperstable liposomes is correlated with in vitro and in vivo killing of cancer cells. In one example, xenograft mice treated with a single dose of hyperstable liposomal BI 2536 show tumor volume reduction lasting 12 days and complete responses in 20% of treated mice. Treatment with two doses weekly apart increases the response rate to 75% of treated mice.

Unless otherwise indicated, all technical and scientific terms used in this document have the same meaning as generally understood by a person skilled in the art to which the invention pertains.

The term "about" or "approximately" means within a statistically significant range of value. Such a range may be within an order of magnitude, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5% of the set value or range. The tolerance covered by the terms "about" or "approximately" depends on the particular system under study and can be easily estimated by a person skilled in the art.

The term "cancer" as used herein refers to a group of diseases involving abnormal cell growth that can invade or spread to other parts of the body. Cancer includes carcinomas such as glioma, head and neck, kidney, lung, medulloblastoma, melanoma, Merkel's carcinoma, mesothelioma, neuroblastoma, cancer of the esophagus, ovary, pancreas, prostate, stomach, testis, thyroid; leukemias such as acute myeloid leukemia, chronic myeloid leukemia, hairy cell leukemia, lymphoblastic T-cell leukemia, T-cell leukemia, B-cell leukemia; lymphomas such as anaplastic large cell lymphoma, B-cell lymphoma, Burkitt's lymphoma, Hodgkin's lymphoma; and myeloma.

The term "mitosis-inhibiting drug" means a drug that targets mitosis-regulating enzymes such as microtubule-regulating enzymes, Polo-like kinases (PLKs), kinesin spindle protein (KSP), aurora kinases, and the like. The term "anti-mitotic drug" or "anti-mitotic drug" may be used interchangeably with "anti-mitosis drug".

As used herein, a "hyper-stable liposome" refers to a liposome-encapsulated drug having slow drug release due, in part, to the anions and cations present in the interior of the liposome. The slowest release rate for hyperstable liposomes is closely related to the killing of cancer cells.

The term "slow drug release" refers to a quantified drug release from a liposome-encapsulated drug of less than 0.6% in 12 hours or less than 5% in 8 days when the liposomes are suspended in 600 mM sucrose.

In one aspect, the present invention provides hyperstable liposomes encapsulating an anti-mitotic drug. In some embodiments, the anti-mitotic drug is a polo-like kinase inhibitor, such as BI 2536, ON01910, GSK 461364, HMN 214, or BI 6727. In other embodiments, the anti-mitotic drug is a kinesin spindle protein inhibitor, such as Ispinesib (SB 715992 ), SB 743921, MK 0731, or ARRY 520. In some embodiments, the antimitotic agent is an aurora kinase inhibitor such as MK 0457 (VX 680), AZD 1152, PHA 680632, PHA 739358, MLN8054, MLN8237, R763, AT9283, SNS 314, SU 6668, ENMD 2076, BI 811283, CYC116, ENMD 981693, or MKC 1693. In some embodiments, the antimitotic agent is BI 2536 or Ispinesib.

In some embodiments, any suitable liposomal moiety may be used to prepare hyperstable liposomes. In some embodiments, hyperstable liposomes are spatially stabilized. In some embodiments, hyperstable liposomes are derived from a fat blend containing HFPC:Chol:DSPE-PEG2000 (HPC: hydrogenated egg L-α-phosphatidylcholine; Chol: cholesterol; 3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] in a molar ratio of 50:45:5.

In some embodiments, the hyperstable liposomes contain an internal environment having one or more anions, preferably two or more anions, which provide a slow release of the anti-mitotic agent from the hyperstable liposomes. In some embodiments, one or more anions, or preferably two or more anions, may be citrate, acetate, phosphate, 2-(N-morpholino)ethanesulfonate, chloride, citrate and acetate, citrate and 2-(N-morpholino)ethanesulfonate, citrate and chloride, acetate and phosphate, acetate and 2-(N-morpholino)ethanesulfonate, acetate and chloride, phosphate and 2-(N-morpholino)ethanesulfonate, phosphate and chloride, 2-(N-morpholino)ethanesulfonate and chloride. In some embodiments, the chloride is in the form of HCl. In some embodiments, the ratio of the two anions may be from about 1:7 to about 7:1. In other embodiments, the ratio of the two anions may be from about 1:3 to about 3:1. In some embodiments, the anions are citrate:phosphate in a ratio of about 1:3 to about 1:7, preferably about 1:3. In some embodiments, the anions are citrate:acetate in a ratio of about 1:3 to about 3:1, preferably about 1:3. In some embodiments, the internal environment contains one or more cations. In some embodiments, one or more of the cations may be sodium, ammonium, triethylammonium, copper, magnesium, zinc, or iron. The best combinations and ratios of anions and cations can be easily determined for a given anti-mitotic drug experimentally in mice, for example, using the techniques described here.

In some embodiments, the hyperstable liposomes of the present invention may contain one or more of the anions of the present invention in any suitable form, eg, in the form of an acid or a salt containing a polyanion and a cation, preferably as a salt. The amount of the anion may be stoichiometrically equivalent to or different from the amount of the cation. In some embodiments, the hyperstable liposome of the present invention comprises one or more anionic cation salts wherein there is a cation concentration gradient or a pH gradient across the membrane of the liposome. In another embodiment, the hyperstable liposome of the invention comprises one or more ammonium anionic salts of the invention. In yet another embodiment, the hyperstable liposomes of the present invention comprise anions within the hyperstable liposomes, and the anions in the medium containing the hyperstable liposomes are partially or substantially removed by any suitable means known to those skilled in the art, e.g., dilution, ion exchange chromatography, size exclusion chromatography, dialysis, ultrafiltration, absorption, precipitation, etc. In some embodiments, the hyperstable liposome with enclosed anion(s) also has a transmembrane gradient effective to retain substances in the hyperstable liposome. Examples of such transmembrane gradients include a pH gradient, an electrochemical potential gradient, a cationic ion gradient, or a solubility gradient. Methods for creating transmembrane gradients are known in the art of liposomes.

In some embodiments, hyperstable liposomes enter target cells using the porosity of the tumor endothelium. In other embodiments, the hyperstable liposomes of the present invention may also be targeted liposomes, for example, liposomes containing one or more targeted groups or biodistribution modifiers on the surface of the liposomes. The directed group can be any agent that is capable of specifically binding or interacting with the desired agent. In some embodiments, the directed group is a ligand. In some embodiments, the ligand preferentially binds to the taken up cell in which the liposome-enclosed substance exerts the desired effect (target cell). The ligand is usually a member of a binding pair, where the second member is present on or in the target cells or in the tissue containing the target cell. See, for example, US patent No. 8,922,970, included here by reference.

Liposomes in accordance with this invention can be obtained by any suitable method known or later discovered by a person skilled in the art. See, for example, Gregoriadis [25], US patent No. 8,992,970 and US patent No. 9,023,384, each of which is incorporated here by reference. Liposomes are usually produced using various methods in which water soluble (hydrophilic) materials are impregnated by using an aqueous solution of these materials as a hydrogenation liquid or by adding a drug/drug solution at some stage during the production of liposomes. Fat-soluble (lipophilic) materials are solubilized in an organic solution of the constituent liquid and then evaporated to a dry drug containing a fatty film after its hydration. These methods involve loading imprisoned agents before or during production (passive loading). However, certain types of compounds with ionizable groups, and those that exhibit both fat and water solubility, can be introduced into liposomes after the formation of unchanged vesicles (remote or active loading).

When preparing liposomes with a mixed fat composition, the fats are first dissolved and mixed in an organic solvent to obtain a homogeneous mixture of fats. In some embodiments, the organic solvent is chloroform or chloroform:methanol mixtures. Once the fats are thoroughly mixed in the organic solvent, the solvent is removed to leave a fatty film. In some embodiments, the organic solvent is removed by rotary evaporation under reduced pressure to leave a thin film of oil on the sides of the round bottom flask. The fat film is usually thoroughly dried overnight under high vacuum to remove residual organic solvent. Hydration of the dry fat film is accomplished by adding an aqueous buffer solution to the dry fat container and stirring at a temperature above the fat transition temperature. This method produces a population of multilamellar liposomes (MLL) that are heterogeneous in both size and shape (eg, 1-5 µm in diameter). Liposome size reduction techniques such as sonication to form single monolamellar vesicles (OMVs) or extrusion through polycarbonate filters to form large monolamellar vesicles (LMVs). Additional details and other methods for preparing drug-encapsulated liposomes can be found in Fritze et al. [16], Dua et al. [20], Laouini et a. [21], US Pat. No. 8,992,970, and US Pat. No. 9,023,384, each of which is incorporated herein by reference.

In some embodiments, the hyperstable liposomes of the present invention are nanosized using saturated phosphatidylcholine coupled with high cholesterol to reduce membrane permeability. In some embodiments, hyperstable liposomes are further formulated using PEGylation or other conjugation for spatial stabilization. In other embodiments, the saturated phosphatidylcholine may be replaced by other membrane-forming phospholipids. In some embodiments, hyperstable liposomes are prepared using conventional techniques or as described herein. In some embodiments, the membrane-forming phospholipids are saturated phosphatidylcholine (PC), any synthetic phosphatidylcholine (PC) with terminal saturated fatty acids, or membrane-forming fats. In some embodiments, the synthetic PC may be dimyristoyl-phosphatidylcholine, dipalmitoyl-phosphatidylcholine, or distearoyl-phosphatidylcholine. In some embodiments, the saturated phosphatidylcholine (PC) is hydrogenated egg yolk phosphatidylcholine (HEG). In other embodiments, the membrane-forming fat is saturated sphingomyelin, saturated phosphatidylethanolamine, saturated phosphatidylglycerol, saturated phosphatidylinositol, or saturated phosphatidylserine.

In some embodiments, the conjugate may be polyethylene glycol, polypropylene glycol, polybutylene glycol, or a copolymer of polyalkylene glycols such as a block copolymer of polyethylene glycol and polypropylene glycol), dextran, pullulan, ficol, polyvinyl alcohol, styrene-maleic anhydride alternating copolymers, divinyl ether-maleic anhydride alternating copolymers, amylose, amylopectin, chitosan, mannan, cyclodextrin, pectin or carrageenan. In some embodiments, polyethylene glycol (PEG) is used as the conjugate (K-PEG). In some embodiments, the PEG has a molecular weight of about 500 to about 10,000, preferably about 1,000 to about 5,000, more preferably about 2,000.

In some embodiments, the PEG or other conjugate is conjugated with distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPFE), dimyristoylphosphatidylethanolamine (DMPE), distearoylglycerol (DSG), dimyristoylglycerol (DMG), cholesteryl conjugate, stetaryl conjugate (STR), C8 conjugate ceramide. In some embodiments, the conjugate is PEG2000 and the conjugate is DSFE-PEG2000, DPFE-PEG2000, DMPE-PEG2000, DSG-PEG2000, DMG-PEG2000, cholesteryl-PEG2000, CSTR-PEG2000, C8 ceramide-PEG2000 or C16 ceramide0.

In some embodiments, sterically stabilized liposomes are prepared from a PC:cholesterol:C-PEG formulation in which the molar ratio of PC:cholesterol is typically 2:1-1:1 with C-PEG typically present at 5% (mol/mol ). In some embodiments, the PC:cholesterol:C-PEG formulation has a molar ratio of 50:45:5. In some embodiments, the formulation is HFPC:cholesterol:DSPE-PEG2000. In some embodiments, the HFPC:cholesterol:DSPE-PEG2000 formulation has a molar ratio of 50:45:5.

In some embodiments, liposomes are prepared by solubilizing the formulation described herein in chloroform. This solution is dried to a thin film by rotary evaporation and then in vacuo overnight. The film is hydrated with a hydration buffer containing the desired saline solution, such as described herein, as the internal medium of the liposome and immersed in a water bath sonicator. The mixture of liposomes is first sonicated and then extruded to form OMB. In some embodiments, the OMF is dialyzed against sucrose to change the environment of the liposomes.

In some embodiments, the mitosis-inhibiting drug is actively loaded into liposomes via a pH gradient method well known in the art. In some embodiments, the mitosis-inhibiting drug is first coated in a thin film in a suitable container and then dried. In some embodiments, the liposomes are loaded at a 3:1 fat:drug concentration and diluted to that desired concentration with water. The mixture is then incubated in a high temperature water bath to enhance loading and then dialyzed on sucrose to remove unencapsulated drug.

In some embodiments, hyperstable liposomes in accordance with this invention are obtained as follows. The fat mixture HAPC:Hol:DSFE-PEG2000 in a molar ratio of 50:45:5 is dissolved in chloroform. The mixture is dried to a thin fat film in a round bottom flask by rotary evaporation and then dried under high vacuum overnight, then hydrogenated with the desired brine as the internal medium of the liposome. The resulting 100 mM fat slurry was sonicated on a sonicator bath for 1 hour and then extruded ten times using a Lipex Thermobarrel Extruder through double folded 100 nm Nuclepore filters to form single monolamellar vesicles (OMVs). These OMB dialyzed in 300 mm sucrose at 4°C with three changes of fresh sucrose solution for 24 hours to replace the external environment of the liposomes. Liposomes are stored in glass tubes at 4° C. until intended use.

In one example, the mitosis inhibitory drug BI 2536 is actively loaded into liposomes via a pH gradient method. BI 2536 is first coated as a thin film in a scintillation tube by dissolving in ethanol, and then dried by rotary evaporation. The BI 2536 film is then vacuum dried for at least 24 hours. Liposomes are loaded at a 3:1 fat:drug concentration and diluted to a final concentration of about 50 to about 70 mM fat with water. The mixture is then incubated in a 70° C. water bath to enhance loading and then dialyzed in 300 mM sucrose for at least 36 h to remove unencapsulated BI 2536. After dialysis, the liposomes are stored in glass tubes until use.

In some embodiments, the hyperstable liposomes of the present invention are sufficiently storage stable, for example, as measured by the percentage of entrapped substance released outside the hyperstable liposomes or still retained within the hyperstable liposomes after a certain period of time from the initial loading of the substance into the hyperstable liposomes according to with this invention. For example, the composition of the hyperstable liposome in accordance with this invention is stable at 4°C for at least 6 months.

Preferably, the liposome-encapsulated anti-mitotic agent remains encapsulated in the liposome until the hyperstable liposome reaches the site of intended action, for example, in the case of a patient-administered liposomal anti-mitotic drug, a tumor. The hyperstable liposomes of the present invention exhibit unexpected stability against release (leakage) of an entrapped anti-mitotic drug under in vivo conditions , such as in mammalian blood. Notably, the hyperstable liposomes of the present invention, having such a low in vivo circulating drug release rate, showed significant in vitro antitumor activity. The hyperstable liposomes of the present invention provide an unexpected combination of high potency of entrapped antimitotic drugs and low toxicity.

In some embodiments, a liposomal composition is provided that contains the hyperstable liposomes described herein in an aqueous medium. In some embodiments, hyperstable liposomes have an internal water space separated from the aqueous medium by a membrane. In some embodiments, the membrane contains 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], hydrogenated egg L-α-phosphatidylcholine, and cholesterol. In some embodiments, the anti-mitotic drug, anion(s) and cation(s) are enclosed within the hyperstable liposomes, wherein the anti-mitotic drug enclosed within the hyperstable liposomes has a concentration greater than the concentration of the anti-mitotic drug in an aqueous medium.

In some embodiments, a pharmaceutical composition is provided that contains the hyperstable liposomes described herein, with or without at least one pharmaceutically acceptable excipient and/or carrier. In some embodiments, pharmaceutically acceptable carriers are normal saline, isotonic dextrose, isotonic sucrose, Ringer's solution, and Hank's solution. A buffer may be added to obtain an optimum pH for storage stability. For example, a pH of about 6.0 to about 7.5, more preferably a pH of about 6.5, is optimal for lipid stability of the liposome membrane and provides excellent retention of entrapped substances. Histidine, hydroxyethyl piperazine ethyl sulfonate (HEPES), morpholipoethyl sulfonate (MES), succinate, tartrate and citrate, usually at a concentration of 2-20 mM, are typical buffer substances. Other suitable carriers include, for example, water, buffered aqueous solution, 0.4% NaCl, 0.3% glycine, and the like. Protein, carbohydrate, or polymeric stabilizers and tonicity correctors, such as gelatin, albumin, or polyvinylpyrrolidone, may be added. The tonicity of the composition can be adjusted to a physiological level of 0.25-0.35 mol/kg with glucose or a more inert compound such as lactose, sucrose, mannitol or dextrin. These compositions can be sterilized by conventional well known sterilization methods, for example by filtration. The resulting aqueous solutions can be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation is combined with a sterile aqueous medium prior to administration.

In some embodiments, pharmaceutically acceptable excipients may be used on demand to approximately physiological conditions, such as pH adjusters and buffering agents, tonicity adjusting agents, and the like, e.g., sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc. d. Additionally, the hyperstable liposome suspension may include fat protecting agents that protect fats from free radical and fat oxidative damage during storage. Lipophilic free radical scavengers such as alpha-tocopherol and water-soluble iron-specific chelators such as ferrioxamine are suitable.

The concentration of hyperstable liposomes according to the invention in pharmaceutical compositions can vary widely, i. e. from less than about 0.05% usually or at least about 2-10% to at least 30-50% by weight, and it is chosen mainly by volume of liquid, viscosity, etc., in accordance with the particular method of administration chosen. For example, the concentration may be increased to reduce the fluid load associated with treatment. This may be particularly desirable in patients with atherosclerosis-related congestive heart failure or severe hypertension. Alternatively, pharmaceutical compositions consisting of irritating fats may be diluted to low concentrations to reduce inflammation at the injection site.

In a second aspect, the present invention provides a method for treating cancer using the hyperstable liposomes described herein. In some embodiments, the amount of hyperstable liposome pharmaceutical composition administered depends on the particular anti-mitotic drug contained within the hyperstable liposomes, the cancer being treated, the type of hyperstable liposomes used, and the judgment of the attending physician. In general, the amount of hyperstable liposome pharmaceutical composition administered should be sufficient to deliver a therapeutically effective dose of the particular anti-mitotic drug.

The amount of the hyperstable liposome pharmaceutical composition required to deliver a therapeutically effective dose can be determined by conventional in vitro and in vivo methods known in the art of drug testing. See, for example, Budman et al. [22]. Therapeutically effective doses of various antimitotic drugs are well known to those skilled in the art; and in accordance with the present invention, an anti-mitotic drug delivered through a pharmaceutical composition in accordance with the present invention provides at least the same or 2-fold, 4-fold or 10-fold activity compared to the activity obtained by administering the same the amount of anti-mitotic drug in a generally non-liposomal formulation. Typically, dosages of the hyperstable liposome pharmaceutical composition of the present invention range from about 0.005 to about 500 mg of therapeutic agent per kilogram of body weight, most commonly from about 0.1 to about 100 mg of therapeutic agent/kg of body weight.

Typically, the pharmaceutical composition according to the invention is prepared as a topical or injectable formulation, either in liquid solution or suspension. However, solid forms suitable for dissolution in or suspension in liquid vehicles prior to injection can also be prepared. The composition may also be formulated as an enteric coated tablet or gel capsule by methods known in the art.

The hyperstable liposome composition of the present invention may be administered by any route that is medically acceptable, which may depend on the cancer being treated. Possible routes of administration include injection, parenteral routes such as intramuscular, subcutaneous, intravenous, intraarterial, intraperitoneal, intraarticular, epidural, and intrathecal or others, as well as oral, nasal, ocular, rectal, vaginal, topical, or pulmonary, such as inhalation. For the delivery of antimitotic drugs formulated in liposomes according to the present invention to tumors of the central nervous system, slow, continuous intracranial infusion of liposomes directly into the tumor (convection delivery or KD) is particularly preferred. See Saito et al. [23] and Mamot et al. [24]. The compositions can also be directly applied to the surface of fabrics. Administration with delayed release, pH-dependent release, or other specific, chemically or environmentally mediated release is also specifically included in the invention, such as depot injections or erodible implants, for example.

As shown in the following examples, a combinatorial anionic diversity approach is described for the identification of slowly releasing hyperstable liposomal formulations. Although the examples focus on the citrate:phosphate anion pair, other hyperstable anion pairs found in the screen (Figure 4) also give similar results for BI 2536. If the citrate:phosphate pair is changed to citrate:acetate, mice treated with a single dose of this alternative version showed the same efficacy at a citrate:acetate ratio of 1:3, giving the greatest tumor reduction (FIG. 8A). A natural means of adding even more variety is to change the cationic identity. For example, replacing the sodium cation with ammonium for the citrate:acetate pair significantly increased the rate of tumor regression (FIG. 8B).

Hyperstable liposomes solve two conceptual problems. Extending the temporal availability allows antimitotic drugs to capture more tumor cells in the replication process. In addition, the low concentration of remaining drug achieved by hyperstable liposomes is less likely to cause mitotic slippage [19]. This means that fewer tumor cells should escape the intended effect of the drug. This approach can be applied to any antimitotic drugs, not just BI 2536. Therefore, hyperstable encapsulation has the potential to revive the clinical utility of the 24 drugs that fall into this class. One advantage of hyperstable liposomes is the prolongation of bioavailability over a two-week time frame for a single dose, allowing clinicians to achieve higher efficacy with less frequent dosing. Another advantage is the absence of irreversible neuropathy after treatment with hyperstable liposomes compared to other drug classes, which is an attractive feature of mitosis inhibitors in terms of toxicity. The description of the general mode of reactivation of unsuccessful inhibitors of mitosis opens the door to many possibilities and demonstrates that the idea of mitosis inhibition is not untenable; delivery matters.

EXAMPLES

The present invention has been described with reference to the following examples, which are provided by way of illustration and are not intended to limit the invention in any way. Were used standard techniques well known in the art, or techniques specifically described below.

EXAMPLE 1

Materials and methods

Liposome preparation: 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) and hydrogenated egg L-α-phosphatidylcholine (HEPQ) are purchased from Lipoid, and cholesterol ( Hol) are purchased from Sigma-Aldrich. The fat mixture HAPC:Hol:DSFE-PEG2000 in a molar ratio of 50:45:5 was dissolved in chloroform (Takara). The mixture is dried to a thin fat film in a round bottom flask with rotary evaporation (Eyela NVC-2200/N-1100) and then dried under high vacuum overnight, then hydrogenated with the desired saline solution as the internal medium of the liposome. The resulting 100 mM fat slurry was sonicated with a bath sonicator (S30H Elmasonic) for 1 hour and then extruded ten times using a Lipex Thermobarrel Extruder (Northern Lipids) through double-folded 100 nm Nuclepore filters (Whatman) to form single monolamellar vesicles (SMVs). ). These OMB dialyzed in 300 mm sucrose (Sigma) at 4°C with three changes of fresh sucrose solution for 24 hours to exchange the external environment of the liposomes. Liposomes are stored in glass tubes at 4° C. until intended use.

Loading BI2536 into liposomes: BI 2536 (Axon) is actively loaded into liposomes in a pH gradient manner. The desired BI 2536 is first coated with a thin film in a scintillation tube by dissolving in ethanol followed by rotary evaporation drying. The BI 2536 film is then vacuum dried for at least 24 hours. The liposomes are loaded at a 3:1 fat:drug concentration and diluted to a final concentration of 50-75 mM fat with water. The mixture is then incubated in a 70°C water bath to speed up loading and then dialyzed in 300 mM sucrose for at least 36 h to remove unencapsulated BI 2536. After dialysis, the liposomes are stored in glass tubes until use, and a portion of the sucrose dialysate is stored at 4 °C for subsequent determination of encapsulation efficiency.

Determination of BI2536 Encapsulation: The amount of BI 2536 loaded into liposomes is determined by direct calculation (in vitro studies) or inverse calculation (for animal studies). For direct calculation, 1 μl of liposomes are diluted with 20 μl of ethanol and read via fluorometric measurement using 360 nm excitation and 470 nm emission (Tecan Infinite M200). The amount of BI2536 is determined by comparison with a standard curve. For back calculation, 100 µl of 1-nonanol (Merck) is used to extract the encapsulated BI 2536 from 1.5 ml of dialysate, vortexed for 1 h. 330 nm excitation and 370 nm emission (Tecan Infinite M200). The concentration of BI 2536 in dialysates is determined by comparison with a standard curve, and the encapsulation efficiency is then calculated by the formula

where A=[BI 2536 in dialysate ] _{without loaded drug} and B=[BI 2536 in dialysate ] _{of the sample} .

Cell culture: HCT116 (CCL-247, human colorectal carcinoma) was purchased from American Type Culture Collection (ATCC) and cultured using McCoy's 5A Medium (Life Technology) supplemented with 10% fetal calf serum (Thermo Scientific). Cells are incubated at 37°C with 5% CO ₂ and subcultured every 2-3 days when the confluence reaches ~80%.

Determination of EC ₅₀ : Approximately 7×10 ³ HCT116 cells are seeded in 96-well plates, reaching ~50% confluence after overnight incubation. The media in the wells are replaced with fresh media supplemented with either free BI 2536 or liposomal BI 2536. Concentrations of applied BI 2536 are created by serial dilutions of 1 μM BI 2536. Wells containing media alone are used as controls. At least 3 repetitions are carried out for each composition. SYBR Green I (Life Technologies) is used to quantify DNA as an indicator of cell survival. It is carried out first by incubating the cells with 50 μl of 0.2% sodium dodecyl sulfate at 37°C for 2 hours to destroy them. 150 µl of SYBR Green solution (1:750 dilution in water) is then added to each well and the fluorescence intensity is measured (ex: 497 nm/ex: 520 nm) using a Tecan plate reader. Fluorescence intensity values are entered into GraphPad Prism V5. Logistic regression curves and EC ₅₀ are determined by setting the highest fluorescence value as 100% survival and the lowest fluorescence value as 0% survival.

Animal Studies: All animal studies are approved by the Institutional Animal Care and Use Committee of Temasek Life Sciences Laboratory and National University of Singapore (NUS). Female NCr nude mice (5-8 weeks old) were purchased from (Singapore/InVivos) and xenografted HCT116 cells were injected subcutaneously. HCT116 cells were grown as described above in 600 cm ² dishes (Corning) and each dish was transplanted into 5 mice when confluence reached ~80%.

Efficacy Studies: Free BI 2536 (dissolved in 0.1 N HCl, saline) or indicated liposomal formulations of BI 2536 are administered by slow tail vein injection 7 days after HCT116 transplantation. Tumor volumes were at least 150 mm ³ and were calculated as length×width ² ×0.5. All measurements were made using a vernier caliper and the mice were weighed every other day. Mice are hydrated subcutaneously with 1 ml of Hartmann's solution daily for 5 days after treatment to ensure mice are fully hydrated.

Pharmacokinetic Study: Mice bearing HCT116 xenografts are treated with the indicated free BI 2536 or BI 2536 liposomal formulations and are sacrificed at the indicated time points post-treatment to harvest heart, tumor, muscle, kidney, liver and spleen. Organs are weighed and stored at -80°C until tissue processing. Tissues are treated by immersion in chaotropic 8M urea (Vivantis) and homogenized in a Bertin Homogenizer using 0.5 mm diameter zirconium beads (Biospec). Homogenized tissues are centrifuged at maximum speed in a bench top centrifuge for one hour and 800 µl of supernatant is collected for BI 2536 extraction using 100 µl of nonanol and gentle rotation for 1 hour. The nonanol and sucrose phases are separated by rapid centrifugation, and 20 μl of the nonanol layer is read by fluorometric measurement (ex: 330 nm/ex: 370 nm) using a Tecan plate reader. BI2536 is quantified by comparison with a standard curve and then normalized to tissue weight.

Histology: Mice are sacrificed to collect tumor tissue on the indicated days after treatment. Tumor tissues were frozen in OCT medium (Sakura Finetek) and stored at -80° C. until sectioned. 10 μm sections of tumor tissue were obtained using a CM3050S cryostat (Leica). Tissue sections were fixed in methanol and immediately stained with Hematoxylin and Eosin (H&E). To perform H&E staining, tumor sections are first overstained with filtered Harris solution (Sigma), washed with running tap water, immersed in acid-alcohol (1% hydrochloric acid, 70% ethanol), and then washed with tap water. The tissue sections are then immersed in 0.2% ammonia water (Sigma) until blue. After rinsing with tap water for 10 minutes, tissue sections are stained with eosin-phloxin (Sigma and Merck, respectively) and immersed in 95% ethanol to wash out excess stain. The tissue sections are dried overnight, then equipped with Permount (Fisher). All H&E stained sections are viewed and bright field imaged using an Axioplan 2 microscope (Carl Zeiss, Inc.) equipped with a DXM 1200F camera (Nikon) and a 63X objective.

Preparation of BI2536 in buffers: BI 2536 is first dissolved in ethanol and then coated in 1.5 ml microcentrifuge tubes by spin drying. The BI 2636 coated tubes are then further dried overnight under high vacuum, then resuspended in the indicated saline solutions to give a final concentration of 500 μM. In order to completely dissolve the coated BI2536, the tubes are rapidly vortexed and sonicated in a bath for 1 minute, then used for analysis.

Extraction with hexanol: BI 2536 from 1 ml of the indicated buffer is extracted with 100 μl of 1-hexanol (Merck) with rapid shaking for 1 hour. Hexanol and buffer layers are separated by rapid centrifugation, and 20 μl of the hexanol layer is analyzed for fluorescence intensity using a Tecan plate reader (ex: 330 nm, ex: 370 nm).

Liposome stability assay: Fluorescence dequenching of leaked BI 2536 is used as an indicator of liposome instability. All fluorescence readings were performed using a Tecan plate reader (ex: 280 nm, ex: 385 nm). Triton-X100 (sigma) is added to achieve a final concentration of 0.2% for complete isolation of BI2536 from liposomes and fluorescence readings are performed again. To calculate the fraction of BI 2536 recovered, the fluorescence data before the addition of triton is divided by the fluorescence data after the addition of triton. For stability measurements, the liposome formulations are diluted 50× with either water or 600 mM sucrose solution and fluorescence is measured using a Tecan plate reader at the start and after 12 hours of incubation at 37°C using a Tecan plate reader. To determine long-term stability, liposomes are stored in a 37° C. incubator after dilution in water or 600 mM sucrose, and fluorescence readings are performed on the same days indicated.

EXAMPLE 2

Liposomal BI 2536 release rates are inversely correlated with tumor cell kill

The degree to which anion species and concentration can affect the physicochemical properties of BI 2536 is studied using the following solutions adjusted to pH 3: sodium citrate (C), sodium acetate (A), sodium phosphate (P), 2-(N -morpholino)ethanesulfonic acid (M); and hydrochloric acid (H). The tendency of BI 2536 to release to hexanol from these solutions was measured at various concentrations (FIG. 1). It was found that the type of anion affects the efficiency of extraction with hexanol, and that this efficiency either increases (P, A) or decreases (M, C, H) in an anion concentration dependent manner (FIG. 2). It has also been found that the diversity of these hexanol extraction efficiency curves can be further increased by using paired combinations of these anions (FIG. 3). Based on these results, it was concluded that these anions could be used in a combinatorial manner to create a library of liposomal formulations with different release rates.

To identify hyperstable forms with slow release of liposomal BI 2536, liposomal encapsulated variants of all 15 single and double anion combinations were obtained and BI 2536 was remotely loaded into their interior. The fluorescence of BI 2536 is quenched when encapsulated at high concentrations in a liposome. Therefore, the release of BI 2536 from liposomes can be measured by the increase in fluorescence due to dequenching. Using this method, BI 2536 leakage for each formulation was measured under hypertonic (600 mM sucrose) and hypotonic (pure water) conditions over time (FIG. 4). As expected, there is a range of release rates from fast (A, H, AH) to slow (all combinations with citrate). The ranking order of these release rates does not differ markedly between hypertonic and hypotonic environments, indicating that it is the liposomal internal environment that determines the drug release rates for liposomal BI 2536 and not external osmotic stress. To investigate whether hyperstable slow release liposomes correlate with cancer cell killing, HCT116 colorectal cancer cells are incubated with serial dilutions of various liposome formulations and their EC ₅₀ values are calculated as a measure of efficacy. Consistent with the hypothesis that hyperstability correlates with cytotoxicity, a high correlation was found between release rates and EC ₅₀ . On the contrary, it was found that there is no similar correlation when the same experiment is carried out using doxorubicin (FIG. 4). This finding is consistent with the idea that while mitotic inhibitors may benefit from hyperstability, the opposite would be true for other drug classes where slow release would not be an advantage.

EXAMPLE 3

Tuning release rate and in vivo efficiency through anion ratios

Since the two anions with the lowest release rate were pure citrate and a combination of citrate and phosphate (FIG. 4), the change in the citrate:phosphate ratio was examined to determine if this had a significant effect on efficiency. Formulations having ratios of 0:1, 1:3, 1:1, 3:1 and 1:0 are tested by the method described above for release rate and EC ₅₀ . The same trend was consistently observed with release rates and EC ₅₀ whether these variables were measured on day 3 or 8 of the cellular cytotoxicity assay (FIG. 5A). It is interesting to note that the slowest release was achieved at a citrate:phosphate ratio of 1:3, indicating that this combinatorial ratio was synergistic and not just an average result of pure citrate and pure phosphate. This result also suggests that it is important to identify the optimal citrate:phosphate ratio in order to maximize in vivo efficacy in actual solid tumors. To identify this ratio, mice with established human rectal and colon xenografts are treated with liposomes having citrate:phosphate ratios of 1:7, 1:4, 1:3, 1:2, and 1:0.5. Consistent with the previously obtained 1:3 ratio, the best in vivo efficacy was observed at 1:3 and 1:4 citrate:phosphate ratios (FIG. 5B). Importantly, the reduction in tumor volumes for these ratios continued for two weeks, and this observation is consistent with the expected slow release from these hyperstable liposomes. Although the 1:4 ratio gave a greater antitumor effect than the 1:3 ratio, it also resulted in greater weight loss. Therefore, 1:3 is accepted as the optimal ratio for subsequent experiments. BI 2536 encapsulated in this manner is referred to as "hyperstable".

EXAMPLE 4

Hyperstable Liposomal BI 2536 Induces Complete Response in Mice

Nude mice bearing HCT116 tumor xenografts are treated with a single intravenous injection of hyperstable liposomal BI 2536 with either citrate alone or phosphate alone as the anion. Free unencapsulated BI 2536 is used as a control. Xenografts are treated with reduced volume hyperstable liposomes for 12 days, replicating the prolonged therapeutic effect observed in our previous animal experiments (FIG. 6A). In comparison, liposomes using only citrate or phosphate were indistinguishable from the free drug, demonstrating that the combination of anions produces a synergistic effect that is not present for any pure anion. Mice treated with hyperstable BI 2536 had higher post-treatment body weights and this trend is consistent with the lower toxicity that would be expected with prolonged release of the drug. Importantly, hyperstable BI 2536 significantly improved mouse survival, with complete responses observed in two out of ten mice (Table 1). No complete responses were observed in the other experimental groups.

TABLE 1

Table of complete responses for different types of treatment

Treatment Complete answers One dose Two doses free drug 0/10 0/8 C:F (1:0) 0/10 1/8 C:F (0:1) 0/10 0/10 C:F (1:3) 2/10 6/8

When the same experiment is repeated with two treatment doses 7 days apart instead of a single dose of hyperstable liposomes, the therapeutic effect is extended for an even longer period (Figure 6B) and produces complete responses in 75% of mice (Table 1). Conversely, there were no complete responses in the other experimental groups. The hyperstable liposomes were not only more effective, but also well tolerated, while all other experimental groups showed toxicity after treatment.

EXAMPLE 5

Hyperstable liposomes prolong tumor presence and potency of BI 2536

A reasonable explanation for the improved potency observed with hyperstable liposomes is that the half-life of the drug is improved compared to conventional PEGylated liposomes. Nude mice treated with a single dose of hyperstable liposomes showed significantly higher tumor concentrations of BI 2536 compared to control liposomes containing either pure citrate or pure phosphate (FIG. 7A; Table 2). This trend persisted throughout the 9.5 day measurement period, after which the decrease in tumor volumes made tissue processing inappropriate. The impact of hyperstable liposomal BI 2536 on the tumor (measured by area under the curve) was 5 times higher compared to citrate liposomes and 3 times higher compared to phosphate liposomes. Drug concentrations in healthy tissues (spleen, muscle, kidney, heart, and liver) were also increased for hyperstable liposomes, although this trend was not statistically significant at 10 hours (FIG. 7A; Table 2). Despite these higher tissue concentrations, the hyperstable liposomes were less toxic than the control liposomes, suggesting that most of the BI 2536 in the hyperstable liposome remains safely encapsulated as it passes through healthy tissues in the blood. Taken together with an overall increase in area under the curve, the data suggest that hyperstable encapsulation increases the circulating half-life of BI 2536, thereby increasing perfusion and retention of BI 2536 in the tumor compartment (Fig. 7B). The hallmark of BI 2536 (or any antimitotic chemotherapy) is its ability to inhibit mitotic division in tumors. To investigate whether the higher bioavailability of BI 2536 could explain the difference between hyperstable liposomes and controls, histological analysis of xenograft tumor samples was performed 1.5 and 5.5 days after a single treatment dose (Figures 7C and 7D). On day 1.5, all liposomal formulations of BI 2536 and encapsulated free BI 2536 were associated with mitotic figures observed in approximately 25% of tumor nuclei in histological sections. However, by day 5.5, hyperstable liposomal BI 2536 was associated with a significantly higher proportion of mitotically arrested cells compared to control liposomes and free drug. This extended temporal bioavailability is thought to account for the increased efficacy of hyperstably encapsulated BI 2536.

TABLE 2

p -values (two-sided non-uniform variance test) comparing tissue concentrations of BI 2536 after treatment with hyperstable liposomes (C:P=1:3) and other treatments (C:P=1:0 and C:P=0:1)

Time (h) p values (two-tailed test of non-uniform variance) in relation to treatment Lip_C:F(1:3) Tumor Spleen muscles kidneys Heart Liver C:F(1:0) C:F(0:1) C:F(1:0) C:F(0:1) C:F(1:0) C:F(0:1) C:F(1:0) C:F(0:1) C:F(1:0) C:F(0:1) C:F(1:0) C:F(0:1) 4 0.0457 0.0874 0.0034 0.0019 0.0148 0.0584 0.0016 0.0075 0.0309 0.0345 0.0043 0.0384 eight 0.0173 0.0147 0.0308 0.0521 0.0036 0.0844 0.0009 0.0021 0.0236 0.0237 0.0320 0.0477 sixteen 0.1075 0.1362 0.2486 0.2225 0.2282 0.2219 0.2510 0.2443 0.2611 0.2526 0.2780 0.2600 32 0.1401 0.1876 0.3107 0.3687 0.5529 0.2917 0.2772 0.3765 0.2992 0.3080 0.9983 0.5652 84 0.0028 0.0132 0.2116 0.3501 0.1480 0.2395 0.2550 0.2903 0.3188 0.1696 0.0565 0.1395 132 0.2336 0.2621 0.6901 0.9838 0.2165 0.1886 0.2886 0.3148 0.0878 0.0374 0.5497 0.3018 180 0.0179 0.0220 0.8185 0.0990 0.1551 0.1585 0.3312 0.4393 0.0053 0.0166 0.0455 0.8493 228 0.1084 0.1148 0.4010 0.8696 0.8303 0.8053 0.8389 0.8251 0.0651 0.7096 0.3052 0.4443

The use of the terms "a" and "an" and "the" and similar references in the context of the description of the invention (especially in the context of the following claims) is to be construed as embracing both the singular and the plural, unless otherwise indicated herein or expressly contradicted. context. The terms "comprising", "having", "including", and "comprising" are to be construed as open terms (i.e., meaning "including but not limited to") unless otherwise noted. The listing of ranges of values in this document is simply intended to serve as a concise way of individually referring to each individual value falling within that range, unless otherwise noted, and each individual value is included in the description as if it were separately specified here. All methods described herein may be performed in any suitable order, unless otherwise specified herein or clearly contradicts the context. The use of any and all examples or exemplary language (eg, "such as") provided herein is merely intended to better illuminate the invention and is not meant to limit the scope of the invention unless otherwise stated. No language in the specification should be construed as indicating any unclaimed element as essential to the practice of the invention.

Embodiments of this invention are described herein, including the best method known to the inventors for carrying out the invention. Variations to these embodiments may become apparent to those skilled in the art upon reading the foregoing description. The inventors expect those skilled in the art to use such variations as the case may be, and the inventors expect the invention to be practiced otherwise than specifically described herein. Therefore, this invention includes all modifications and equivalents of the subject matter set forth in the appended claims, as permitted by applicable law. Moreover, any combination of the above elements in all possible cases is covered by the invention, unless otherwise indicated here or otherwise clearly contradicts the context.

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Claims (18)

1. A hyperstable liposome for cancer treatment, containing an internal environment separated from the external environment by a membrane, where the internal environment contains an anti-mitotic drug, two cations enclosed in the internal environment, where the antimitotic drug is a polo-like kinase inhibitor, where the membrane contains 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), hydrogenated egg L-α- phosphatidylcholine (HFPC), and cholesterol (Chol), where the molar ratio of HFPC:Chol:DSPE-PEG2000 is 50:45:5, where the two anions are citrate and phosphate, citrate and acetate, or acetate and phosphate in a molar ratio of 1:3 or 1:4, and wherein the entrapped antimitotic drug has a slow release rate from the hyperstable liposome that is quantitatively less than 0.6% in 12 hours or less than 5% in 8 days, wherein the hyperstable liposomes are suspended in 600 mM sucrose. 2. The hyperstable liposome of claim 1, wherein the two anions are citrate and phosphate, or citrate and acetate. 3. The hyperstable liposome according to claim 1, wherein the two anions are citrate and phosphate. 4. The hyperstable liposome according to claim 1, 2 or 3, wherein the two anions are present in a 1:3 molar ratio. 5. The hyperstable liposome according to claim 1, 2 or 3, wherein the two anions are present in a 1:4 molar ratio. 6. Hyperstable liposome according to any one of paragraphs. 1-5, where one or more cations are sodium, ammonium, triethylammonium, copper, magnesium, zinc or iron. 7. Hyperstable liposome according to any one of paragraphs. 1-6, where the antimitotic drug is BI 2536, ON 01910, GSK 461364, HMN 214, or BI 6727. 8. The hyperstable liposome of claim 7 wherein the antimitotic drug is BI 2536. 9. The hyperstable liposome of claim 1 wherein the antimitotic drug is BI 2536 and wherein the two anions are citrate and phosphate in a 1:3 molar ratio. 10. Liposomal composition for the treatment of cancer, containing a hyperstable liposome according to any one of paragraphs. 1-9 in the aquatic environment. 11. Pharmaceutical composition for the treatment of cancer, containing a hyperstable liposome according to any one of paragraphs. 1-9 and a pharmaceutically acceptable excipient. 12. A pharmaceutical composition for the treatment of cancer according to claim 11, wherein the composition further comprises at least one pharmaceutically acceptable carrier. 13. Hyperstable liposome according to any one of paragraphs. 1-9 for use as a drug for the treatment of cancer in a subject. 14. The use of a hyperstable liposome according to any one of paragraphs. 1-9 in the preparation of a medicament for the treatment of cancer in a subject. 15. Hyperstable liposome according to any one of paragraphs. 1-9 or a liposomal composition according to claim 10 or a pharmaceutical composition according to claim 11 or 12 for use in a method of treating cancer in a subject.

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